ELISA Kit for serodiagnosis of Bovine coronavirus

Diarrhoea is one of the leading causes of death in young calves under one month old. Since Mebus’s 1969 discovery that viruses could be detected in the faeces of calves with diarrheoa, it has been proven that coronavirus can infect the calve and cause sometimes severe diarrhoea. Coronavirus is one of the pathogens associated with gastrotenteritis in young calves. Coronavirus is ubiquitous. As a result, most of the animals coming from intensive livestock farms have specific antibodies against this pathogen. The antibodies produced by the cow in response to natural immunisation or vaccination are transmitted to her calf at birth via the colostrum. The colostrum immunoglobulins frequently are not transmitted to the calves correctly (poor quality colostrum, late administration, too small an amount, pre-calving mastitis, etc…). As a result, the calf will be insufficiently protected from infection. The coronavirus ELISA kit enables one to measure the suckling calf’s specific protection against coronavirus. For this, a serum sample must be taken in the first few days after birth when the calf is still protected by the colostrum and has not yet developed active immunity against the virus. However, you must wait at least 24 hours after the first dose of colostrum before taking the control blood sample to allow intestinal resorption of the immunoglobulins to take place. The kit may also be used to test the efficacy of vaccines.

II – PRINCIPLE OF THE TEST

The 96-well microplates have been sensitised by purified Bovine coronavirus. The kit’s user deposits the previously diluted test sera, plasma or colostrum in the microplate’s wells, then adds the conjugate, which is a specific monoclonal antibody against coronavirus coupled to peroxidase. After incubating and washing the preparation, the chromogen (tetramethylbenzidine) is added. This chromogen has the advantage of being more sensitive than the other peroxidase chromogens and not being carcinogenic. The intensity of the colour is inversely proportionate to the sample’s serum titre. Positive and negative control sera are provided with the kit to be able to validate the test results.

III – COMPOSITION OF THE KIT

– Microplates: 96-well microtitration plates. The entire surface of each microplate has been sensitised with purified bovine coronavirus. – Washing solution: one bottle of 20x concentrated washing solution. The solution crystallises spontaneously when cold. If only part of the solution is to be used, bring the bottle to 21°C +/- 3°C until disappearance of all crystals. Mix the solution well and remove the necessary volume. Dilute the buffer 1:20 with distilled or demineralised water. – Dilution buffer: One bottle of 5x colored, concentrated buffer for diluting samples and conjugate. Dilute this concentrated dilution buffer 1:5 with distilled or demineralised water. If a deposit forms at the bottom of the container filter the solution on Whatman filter paper. – Conjugate: One vial of anti-coronavirus-peroxidase conjugate (horseradish peroxidase-labelled anticoronavirus monoclonal antibody). The reagent must be diluted 1:20 with the dilution buffer. – Positive serum: 1 bottle containing the positive serum. Store this reagent between +2°C and +8°C. – Negative serum: 1 bottle containing the negative serum. Store this reagent between +2°C and +8°C. – Single component TMB: One bottle of the chromogen tetramethylbenzidine. Store between +2°C and +8°C. protected from light. This solution is ready to use. – Stopping solution: One bottle of the 1 M phosphoric acid stop solution